Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers.

A previous study using Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) found human plasma contains short (≤300 nt) structured full-length excised linear intron (FLEXI) RNAs with potential to serve as blood-based biomarkers. Here, TGIRT-seq identified >9,000 different FLEXI RNAs in human cell lines, including relatively abundant FLEXIs with cell-type-specific expression patterns. Analysis of public CLIP-seq datasets identified 126 RNA-binding proteins (RBPs) that have binding sites within the region corresponding to the FLEXI or overlapping FLEXI splice sites in pre-mRNAs, including 53 RBPs with binding sites for ≥30 different FLEXIs. These included splicing factors, transcription factors, a chromatin remodeling protein, cellular growth regulators, and proteins with cytoplasmic functions. Analysis of ENCODE datasets identified subsets of these RBPs whose knockdown impacted FLEXI host gene mRNA levels or proximate alternative splicing, indicating functional interactions. Hierarchical clustering identified six subsets of RBPs whose FLEXI binding sites were co-enriched in six subsets of functionally related host genes: AGO1-4 and DICER, including but not limited to agotrons or mirtron pre-miRNAs; DKC1, NOLC1, SMNDC1, and AATF (Apoptosis Antagonizing Transcription Factor), including but not limited to snoRNA-encoding FLEXIs; two subsets of alternative splicing factors; and two subsets that included RBPs with cytoplasmic functions (e.g., LARP4, PABPC4, METAP2, and ZNF622) together with regulatory proteins. Cell fractionation experiments showed cytoplasmic enrichment of FLEXI RNAs with binding sites for RBPs with cytoplasmic functions. The subsets of host genes encoding FLEXIs with binding sites for different subsets of RBPs were co-enriched with non-FLEXI other short and long introns with binding sites for the same RBPs, suggesting overarching mechanisms for coordinately regulating expression of functionally related genes. Our findings identify FLEXIs as a previously unrecognized large class of cellular RNAs and provide a comprehensive roadmap for further analyzing their biological functions and the relationship of their RBPs to cellular regulatory mechanisms.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2.

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

A Novel Macrophage-Activating Gel Improves Healing and Skin Quality After CO2 Laser Resurfacing of the Chest.

BACKGROUND: After laser resurfacing, it is imperative that an appropriate postoperative regimen is followed for optimal wound healing. There is currently no consensus about which agents should be used. OBJECTIVE: To evaluate the safety and efficacy of a novel macrophage-activating gel in a Phase 2B trial to be used after fractionated ablative laser resurfacing of the chest. MATERIALS AND METHODS: Forty-two adults who received fractionated CO2 laser resurfacing of the chest were randomized (active or placebo) for 5 consecutive days after procedure. Skin quality at baseline and follow-up was assessed by a blinded evaluator using the Fitzpatrick-Goldman Wrinkle Scale. Subject satisfaction with skin healing and quality was also assessed. RESULTS: At 28 days according to the Fitzpatrick-Goldman Wrinkle Scale, 85% of subjects achieved an improvement of at least 33% for the active group versus 50% in the placebo group (absolute difference 35%; p = .04). Similarly, 75% of subjects achieved an improvement score of at least 33% in elastosis in the active group versus 35% in the placebo group at 28 days (40% absolute difference; p = .011). CONCLUSION: This study confirms the potent effects of the novel macrophage-activating gel for optimization of skin healing and quality after laser resurfacing of the chest.

Serious Adverse Events With Injectable Fillers: Retrospective Analysis of 7,659 Patient Outcomes.

BACKGROUND: In total, 2.7 million injectable filler treatments were performed in 2019 in the United States. Although generally considered to be a safe treatment modality, adverse events may occur in rare situations. OBJECTIVE: Analyze serious adverse events from injectable filler treatments, including infections, cutaneous necrosis, blindness, or delayed-onset nodule formation, spanning 11 years for 3 board-certified dermatologists and review their incidence, management, and outcomes. MATERIALS AND METHODS: A retrospective analysis was performed of injectable filler treatments spanning 11 years at a multipractitioner outpatient clinic. Serious adverse events were identified, and treatment measures were documented. A literature search was performed to determine recent trends and outcomes for comparison. RESULTS: Between January 2009 and August 2020, 18,013 mL of injectable filler was administered to 7,659 patients. Of the 18,013 mL administered, 74.1% comprised hyaluronic acid derivatives, 19.19% poly-l-lactic acid, and 6.71% calcium hydroxylapatite. Four serious adverse events were identified. Three events were delayed-onset skin nodule formation. One adverse event was related to vascular compromise and subsequent cutaneous necrosis. After appropriate treatment, all adverse events resolved without significant long-term sequelae. CONCLUSION: Serious adverse events associated with injectable fillers, when performed by board-certified dermatologists, are extremely rare and can be successfully managed with appropriate treatment.

A Randomized, Evaluator-Blind, Split-Face Study Evaluating the Safety and Efficacy of Calcium Hydroxylapatite for Jawline Augmentation.

INTRODUCTION: Jawline augmentation with calcium hydroxylapatite has not yet been evaluated in a prospective study with a split-face design. This study aims to perform the first randomized controlled, split-face study on the efficacy and safety of calcium hydroxylapatite for jawline augmentation using the needle and cannula technique. OBJECTIVE: To perform the first randomized controlled, split-face study on the efficacy and safety of calcium hydroxylapatite for jawline augmentation using the needle and cannula technique. MATERIALS AND METHODS: This is a single-site, randomized, evaluator-blind trial enrolling a total of 10 healthy subjects with at least Grade 1 (mild) on a 4-point Jawline Scale. One side of the face was randomized to receive 1 to 2 syringes of calcium hydroxylapatite with lidocaine (total of 3 mL) for correction of wrinkles and folds along the jawline using both the cannula and needle method, and a balancing treatment will be performed 1 month later. Blinded investigator and subject evaluations will be performed immediately after treatment and at the 30-, 60-, and 90-day visits. RESULTS: Ten subjects were enrolled and completed the trial. There was a improvement in the degree of wrinkling and skin sagging in the 4-point Jawline Scale, with an average of a 1.3-point improvement in the scale on the day of treatment and at the Day 30 visit, which remained improved greater than baseline after 3 months as graded by blinded investigators. The Clinician Global Aesthetic Improvement Score for the treated side versus control, as assessed by blinded investigators, demonstrated a improvement with a 2.3-point improvement on the 5-point scale, and by the final visit on Day 90, most patients had a much improved appearance from baseline. CONCLUSION: This study demonstrates that calcium hydroxylapatite is effective and safe for restoration and augmentation of the jawline using the unique needle and cannula technique.

Review of Post-laser-resurfacing Topical Agents for Improved Healing and Cosmesis.

BACKGROUND: Laser resurfacing produces a controlled skin injury, resulting in a wound healing response. This wound healing response allows for collagen remodeling, which improves skin texture and tone. Topical agents are often employed following laser treatments to facilitate recovery. The introduction of newer small-molecule technologies allow for improved recovery and cosmesis. OBJECTIVE: We sought to perform a critical review of the safety and efficacy of newer small-molecule technologies employed following laser resurfacing. METHODS: We performed a PubMed search of the generic name of the following topicals and included literature relevant to laser procedures, with an emphasis on laser resurfacing: thermal spring water, conjugated linolenic acid, vitamin C/vitamin E/ferulic acid serum, tripeptide/hexapeptide technology-containing products, growth factor serum and gel, recombinant human epidermal growth factor ointment and gel, red deer umbilical cord lining mesenchymal stem cell extract cream and serum, silicone-based gel, and microparticulate (1-3, 1-6 beta-glucan) gel. RESULTS: Our search of the PubMed database yielded 62 results, out of which 17 clinical studies were included in this publication. The majority of aforementioned topicals show promise in terms of improving post-resurfacing recovery or cosmesis. CONCLUSION: Clinical data regarding these agents is limited by the number and quality of studies. It is therefore challenging to propose a recommendation supporting any particular topical. We provide our own provider-specific post-laser resurfacing protocols to offer insight regarding new small-molecule technologies.